Devices for Improved Wound Management

ABSTRACT

The present invention provides devices and compositions for the management of infection of topical lesions, each of the devices and compositions containing protonated/acidified nucleic acids either on its surface, or integrated into the device. These modified nucleic acids are effective as bactericidal and/or bacteriostatic agents without regard to the class of bacteria, so are especially useful when diagnosis is difficult or when multiple infectious organisms are present. The antibiotic activity of nucleic acids of the invention is not dependent on either the specific sequence of the nucleic acid or the length of the nucleic acid molecule. The nucleic acids used in the invention are protonated/acidified to give a pH when dissolved in water of less than pH 7 to about 1, more preferably less than pH 4.5 to about 1, and even more preferably less than pH 2 to about 1.

This application is a continuation-in-part of our earlier filedapplication Ser. No. 09/222,009, filed Dec. 30, 1998, to which we claimpriority under 35 U.S.C. §120 and which is incorporated herein byreference in its entirety.

FIELD OF THE INVENTION

This invention relates generally to devices for promoting closure,healing, and/or prevention of infection in burn wounds, ulcers, donorsites, bites and other shallow wounds. More particularly, this inventionpertains to such devices that have a broad spectrum antibiotic effect.

BACKGROUND OF THE INVENTION

The skin is an essential component of the nonspecific immune system andfunctions as a first barrier to pathogenic infection. Breaches in theskin, i.e., wounds, predispose the patient to infection. Thermal burnscause massive destruction of the integument as well as suppression ofhumoral and cellular immunity, enabling opportunistic organisms that donot generally infect a healthy person to infect a burn victim, bothtopically and systemically. Scratches, bites and ulcers also causeinfection by allowing the introduction of microorganisms into deeper,susceptible tissues.

Each year, approximately 2.4 million Americans are burned. Physicianstreat about 650,000 of the burn victims, 75,000 of these patientsrequire hospitalization, and 12,000 die of burns. One million peopleeach year sustain substantial temporary or permanent disabilitiesresulting from burn injuries. Infectious complications are the leadingcause of morbidity and mortality in serious burn injury, withapproximately 10,000 patients in the U.S. dying of burn-relatedinfections each year. When skin is damaged or missing due to burns,trauma or toxic injury, the mechanical functions of the skin must bereplaced promptly to provide an environment that will optimize cellularregeneration and minimize the chance for sepsis. During this healingprocess, systemic immunosuppression is induced, placing the patient atgreater risk for infection. Burns also predispose the affected area toinfection by damaging the protective barrier function of the skin, thusallowing the entry and colonization of opportunistic organisms.

An ultimate goal of burn wound management is closure and healing of thewound. One way in which wound healing is promoted is the use ofdressings to cover the wound site. An ideal dressing is one thatflexibly covers the wound site and provides a barrier to infectiousorganisms and an environment that promotes the healing process. Apreferred environment is one similar to the patient's own skin inproviding a moisture retaining, germ resistant covering while possiblystimulating the healing. Although biological dressings, e.g., porcinexenografts, have been the dressing of choice since the early 1960's, newsynthetic dressings are being introduced that have the additionalbenefit of being sterile. Both biological and synthetic dressings serveas temporary coverages for wounds and essentially all provide a barrierthat aids in healing.

Burn wound healing has traditionally been augmented by the use oftopical antibiotics. Currently, the most common antimicrobial agentsused for burn victims are silver sulfadiazene cream, mafenide acetatecream, and silver nitrate, which dramatically decrease the bacterialburden of burn wounds and consequently decrease the rate of infection.These compounds, although effective, have limitations. Silversulfadiazene is often used initially, but its value is often limited bybacterial resistance. Mafenide acetate is more broad spectrum in effectbut it has negative side effects such as metabolic acidosis andhypersensitivity.

Other wounds can also be problematic for the treatment of antibioticinfection, especially wounds that are more likely to expose a subject toinfectious agents (e.g., an animal bite), or wounds that are deep and/ordifficult to access (e.g., puncture wounds). Animal bites, includinghuman bites, expose the damaged tissue to a variety of pathogens thatreflect the oral flora of the biting animal. Antibiotic management ofwounds such as animal bites and puncture wounds is thus challenging,since the antibiotics used depend in large part on the potentialpathogens that may have infected the wound. This can be especiallyproblematic in individuals with antibiotic allergies, e.g., penicillinallergies, since treatment for these patients may require combinationtherapies to provide broad spectrum protection. In addition, certainanimal bites, such as snake bites, also can result in severeinflammatory responses and/or tissue necrosis, which renders these bitesespecially prone to infection.

There is a need in the art for devices to promote healing and preventinfection in burns, bites, and other skin lesions that simultaneouslyprovide a physical barrier and a chemical treatment for prevention ofinfection to aid the healing process. There is also a need for sterilemethods of closure that can provide additional antibiotic protection tothese wounds.

SUMMARY OF THE INVENTION

The present invention provides devices and compositions for themanagement of infection of topical wounds and lesions wherein thedevices and compositions are given broad spectrum antibacterialproperties by means of protonated/acidified nucleic acids. Thesemodified nucleic acids may be present on the device surface, orintegrated into the device or composition. Protonated/acidified nucleicacids have broad spectrum activity, i.e., are effective as bactericidaland/or bacteriostatic agents without regard to the class of bacteria, soare especially useful when identification of the infectious agent isdifficult or when multiple infectious organisms are present. The nucleicacids used in the invention are protonated/acidified to give a pH whendissolved in water of less than pH 7 to about 1, more preferably lessthan pH 4.5 to about 1, and even more preferably less than pH 2 to about1.

The nucleic acids of the invention may be protonated/acidified monomersor polymers. Polymers are preferably protonated/acidifiedoligonucleotides from 2-100 nucleotides in length. The nucleic acids ofthe invention may have nuclease resistant backbones, acid resistantbackbones, and, in the preferred embodiment, have both acid resistantand nuclease resistant backbones.

In a first embodiment, the invention provides dressings for wounds whichhave protonated/acidified nucleic acids incorporated into or on thedressing to provide sterility and antibiotic activity. Such dressing maybe comprised of any materials suitable for this use, e.g., polyester oracrylic mesh, and preferably, the dressings are a polyester mesh nettingformed of woven multifilament polyester. Such dressing may also have apolymeric film bonded to one side of the coated solid substrate,preferably of about 0.001 inch+/−about 0.0005 inch.

In another embodiment, the invention provides sutures having a coatingof an effective amount of protonated/acidified nucleic acids. Thepreferred sutures are nonabsorbable, multifilament sutures, preferablypolyester sutures. The protonated/acidified nucleic acid on the sutureis preferably from about 0.1 to about 5 percent of the dry weight of thesuture. The amount used will be an “effective amount” meaning the amountneeded to obtain the desired antibacterial effect over the period oftime the dressing would be expected to be worn.

In yet another embodiment, the invention provides an adhesivecomposition having antibiotic properties for skin contact applications.The concentration of protonated/acidified nucleic acids in said polymercomposition is about 0.1% to about 2% by weight. These adhesives containan adhesive polymer with an effective amount of protonated/acidifiednucleic acid dispersed throughout said polymer. The adhesive ispreferably comprised of an acrylic polymer, and more preferably is amixture of a low molecular weight solid acrylic polymer and a mediummolecular weight solid acrylic polymer. In addition, the adhesivecomposition of the invention preferably has an effective amount of atackifier.

In yet another embodiment, the invention provides wound sealantscomprised of an effective amount of protonated/acidified nucleic acids,preferably about 0.1% to about 2% by weight. The wound sealant containsa fibrinogen activator in a concentration sufficient to initiate clotformation and may also contain fibrinogen and/or platelets. Preferablythe fibrinogen activator is thrombin or batroxobin.

In yet another embodiment, the invention provides a skin substitute withan effective amount of protonated/acidified nucleic acids on itssurface, or which has such modified nucleic acids impregnated into theskin substitute. The protonated/acidified nucleic acids in said skinsubstitute is preferably about 0.1% to about 2% by weight.

It is an object of the invention to provide a sterile environment forhealing of wounds.

It is another object of the invention to provide methods of woundclosure with additional antibiotic properties.

It is yet another object of the invention to prevent bacterial infectionin burn victims.

It is another advantage of the invention that the mechanism of action ofthe protonated/acidified nucleic acids appears to be relativelynon-specific, allowing them to be effective against any bacteriumincluding clinically relevant pathogenic bacteria.

It is another advantage of the invention that the protonated/acidifiednucleic acids are non-toxic to a subject treated with the modifiednucleic acids.

It is a further advantage that the antibacterial effectiveness ofprotonated/acidified nucleic acids is neither length- norsequence-dependent.

It is yet a further advantage that the protonated/acidified nucleicacids of the invention are economical to produce in large quantities,and thus are cost-effective for larger doses.

These and other objects, advantages, and features of the invention willbecome apparent to those persons skilled in the art upon reading thedetails of the antibiotic devices and formulations used in such devicesas more fully described below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph illustrating the establishment of burn wound infectionby subcutaneous and topical administration of Psuedomonas aeruginosa.

FIG. 2 is a graph illustrating the optimization of different routes ofprotonated/acidified oligonucleotide administration for treatment ofburn wound infection.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

Before the present bandages are described, it is to be understood thatthis invention is not limited to particular materials or uses described,as such may, of course, vary. It is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to be limiting, since the scope ofthe present invention will be limited only by the appended claims.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are now described. All publications mentioned herein areincorporated herein by reference to disclose and describe the methodsand/or materials in connection with which the publications are cited.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

DEFINITIONS

The terms “nucleic acid” and “nucleic acid molecule” as usedinterchangeably herein, refer to a molecule comprised of nucleotides,i.e., ribonucleotides, deoxyribonucleotides, or both. The terms includemonomers and polymers of ribonucleotides and deoxyribonucleotides, withthe ribonucleotides and/or deoxyribonucleotides being connectedtogether, in the case of the polymers, via 5′ to 3′ linkages. However,linkages may include any of the linkages known in the nucleic acidsynthesis art including, for example, nucleic acids comprising 5′ to 2′linkages. The nucleotides used in the nucleic acid molecule may benaturally occurring or may be synthetically produced analogues that arecapable of forming base-pair relationships with naturally occurring basepairs. Examples of non-naturally occurring bases that are capable offorming base-pairing relationships include, but are not limited to, azaand deaza pyrimidine analogues, aza and deaza purine analogues, andother heterocyclic base analogues, wherein one or more of the carbon andnitrogen atoms of the purine and pyrimidine rings have been substitutedby heteroatoms, e.g., oxygen, sulfur, selenium, phosphorus, and thelike.

The term “oligonucleotide” as used herein refers to a nucleic acidmolecule comprising from about 2 to about 100 nucleotides, morepreferably from 2 to 80 nucleotides, and even more preferably from about4 to about 35 nucleotides.

The term “monomer” as used herein refers to a nucleic acid molecule andderivatives thereof comprised of a single nucleotide.

The terms “modified oligonucleotide”, “modified monomer”, and “modifiednucleic acid molecule” as used herein refer to nucleic acids with one ormore chemical modifications at the molecular level of the naturalmolecular structures of all or any of the nucleic acid bases, sugarmoieties, intemucleoside phosphate linkages, as well as molecules havingadded substituents, such as diamines, cholesteryl or other lipophilicgroups, or a combination of modifications at these sites. Theinternucleoside phosphate linkages can be phosphodiester,phosphotriester, phosphoramidate, siloxane, carbonate,carboxymethylester, acetamidate, carbamate, thioether, bridgedphosphoramidate, bridged methylene phosphonate, phosphorothioate,methylphosphonate, phosphorodithioate, bridged phosphorothioate and/orsulfone internucleotide linkages, or 3′-3′, 2′-5′, or 5′-5′ linkages,and combinations of such similar linkages (to produce mixed backbonemodified oligonucleotides). The modifications can be internal (single orrepeated) or at the end(s) of the oligonucleotide molecule and caninclude additions to the molecule of the internucleoside phosphatelinkages, such as cholesteryl, diamine compounds with varying numbers ofcarbon residues between amino groups and terminal ribose, deoxyriboseand phosphate modifications which cleave or cross-link to the oppositechains or to associated enzymes or other proteins. Electrophilic groupssuch as ribose-dialdehyde could covalently link with an epsilon aminogroup of the lysyl-residue of such a protein. A nucleophilic group suchas n-ethylmaleimide tethered to an oligomer could covalently attach tothe 5′ end of an mRNA or to another electrophilic site. The termmodified oligonucleotides also includes oligonucleotides comprisingmodifications to the sugar moieties such as 2′-substitutedribonucleotides, or deoxyribonucleotide monomers, any of which areconnected together via 5′ to 3′ linkages. Modified oligonucleotides mayalso be comprised of PNA or morpholino modified backbones where targetspecificity of the sequence is maintained.

The term “nucleic acid backbone” as used herein refers to the structureof the chemical moiety linking nucleotides in a molecule. This mayinclude structures formed from any and all means of chemically linkingnucleotides. A modified backbone as used herein includes modificationsto the chemical linkage between nucleotides, as well as othermodifications that may be used to enhance stability and affinity, suchas modifications to the sugar structure. For example an α-anomer ofdeoxyribose may be used, where the base is inverted with respect to thenatural β-anomer. In a preferred embodiment, the 2′-OH of the sugargroup may be altered to 2′-O-alkyl or 2′-O-alkyl-n(O-alkyl), whichprovides resistance to degradation without comprising affinity.

The terms “acidification” and “protonation/acidification” as usedinterchangeably herein refer to the process by which protons (orpositive hydrogen ions) are added to proton acceptor sites on a nucleicacid. The proton acceptor sites include the amine groups on the basestructures of the nucleic acid and the phosphate of the phosphodiesterlinkages. As the pH is decreased, the number of these acceptor siteswhich are protonated increases, resulting in a more highlyprotonated/acidified nucleic acid.

The term “protonated/acidified nucleic acid” refers to a nucleic acidthat, when dissolved in water at a concentration of approximately 16A₂₆₀ per ml, has a pH lower than physiological pH, i.e., lower thanapproximately pH 7. Modified nucleic acids, nuclease-resistant nucleicacids, and antisense nucleic acids are meant to be encompassed by thisdefinition. Generally, nucleic acids are protonated/acidified by addingprotons to the reactive sites on a nucleic acid, although othermodifications that will decrease the pH of the nucleic acid can also beused and are intended to be encompassed by this term.

The term “end-blocked” as used herein refers to a nucleic acid with achemical modification at the molecular level that prevents thedegradation of selected nucleotides, e.g., by nuclease action. Thischemical modification is positioned such that it protects the integralportion of the nucleic acid, for example the coding region of anantisense oligonucleotide. An end block may be a 3′ end block or a 5′end block. For example, a 3′ end block may be at the 3′-most position ofthe molecule, or it may be internal to the 3′ ends, provided it is 3′ tothe integral sequences of the nucleic acid.

The term “substantially nuclease resistant” refers to nucleic acids thatare resistant to nuclease degradation, as compared to naturallyoccurring or unmodified nucleic acids. Modified nucleic acids of theinvention are at least 1.25 times more resistant to nuclease degradationthan their unmodified counterpart, more preferably at least 2 times moreresistant, even more preferably at least 5 times more resistant, andmost preferably at least 10 times more resistant than their unmodifiedcounterpart. Such substantially nuclease resistant nucleic acidsinclude, but are not limited to, nucleic acids with modified backbonessuch as phosphorothioates, methylphosphonates, ethylphosphotriesters,2′-O-methylphosphorothioates, 2′-O-methyl-p-ethoxy ribonucleotides,2′-O-alkyls, 2′-O-alkyl-n(O-alkyl), 2′-fluoros,2′-deoxy-erythropentofuranosyls, 2′-O-methyl ribonucleosides, methylcarbamates, methyl carbonates, inverted bases (e.g., inverted T's), orchimeric versions of these backbones.

The term “substantially acid resistant” as used herein refers to nucleicacids that are resistant to acid degradation as compared to unmodifiednucleic acids. Typically, the relative acid resistance of a nucleic acidwill be measured by comparing the percent degradation of a resistantnucleic acid with the percent degradation of its unmodified counterpart(i.e., a corresponding nucleic acid with “normal” backbone, bases, andphosphodiester linkages). A nucleic acid that is acid resistant ispreferably at least 1.5 times more resistant to acid degradation, or atleast 2 times more resistant, even more preferably at least 5 times moreresistant, and most preferably at least 10 times more resistant than itsunmodified counterpart.

The term “LD₅₀” as used herein is the dose of an active substance thatwill result in 50 percent lethality in all treated experimental animals.Although this usually refers to invasive administration, such as oral,parenteral, and the like, it may also apply to toxicity using lessinvasive methods of administration, such as topical applications of theactive substance.

The term “alkyl” as used herein refers to a branched or unbranchedsaturated hydrocarbon chain containing 1-6 carbon atoms, such as methyl,ethyl, propyl, tert-butyl, n-hexyl and the like.

The terms “treatment”, “treating” and the like are used herein togenerally mean obtaining a desired pharmacologic and/or physiologiceffect. The effect may be prophylactic in terms of completely orpartially preventing a disease or symptom thereof and/or may betherapeutic in terms of a partial or complete cure for a disease and/oradverse effect attributable to the disease. “Treatment” as used hereincovers any treatment of a disease in a mammal, particularly a human, andincludes:

(a) preventing a bacterial disease from occurring in a subject who maybe predisposed to the disease but has not yet been diagnosed as havingit;

(b) inhibiting a bacterial disease, i.e., arresting its development; or

(c) relieving a bacterial disease, i.e., causing regression and/oramelioration of the disease. The invention is directed toward treatingpatients with any infectious bacteria.

By the term “effective amount” or “therapeutically effective amount” ofprotonated/acidified nucleic acid is meant an amount of aprotonated/acidified nucleic acid sufficient to obtain the desiredphysiological effect, e.g., suppression and/or prevention of bacterialinfection. An effective amount of protonated/acidified nucleic acid isdetermined by the caregiver in each case on the basis of factorsnormally considered by one skilled in the art to determine appropriatedosages, including the age, sex, and weight of the subject to betreated, the condition being treated, and the severity of the wound orinfection being treated.

Nucleic Acid Synthesis

Nucleic acids can be synthesized on commercially purchased DNAsynthesizers from <1 uM to >1 mM scales using standard phosphoramiditechemistry and methods that are well known in the art, such as, forexample, those disclosed in Stec et al., 1984, J. Am. Chem. Soc.106:6077-6089, Stec et al., 1985, J. Org. Chem. 50(20):3908-3913, Stecet al., 1985, J. Chromatog. 326:263-280, LaPlanche et al., 1986, Nuc.Acid. Res. 14(22):9081-9093, and Fasman, 1989, Practical Handbook ofBiochemistry and Molecular Biology, 1989, CRC Press, Boca Raton, Fla.,herein incorporated by reference.

Nucleic acids can be purified by any method known to those in the art.In a preferred embodiment, they are purified by chromatography oncommercially available reverse phase or ion exchange media, e.g., WatersProtein Pak, Pharmacia's Source Q, etc. Peak fractions can be combinedand the samples desalted and concentrated by means of reverse phasechromatography on a poly(styrene-divinylbenzene) based media, such asHamilton's PRP1 or PRP3, or Polymer Labs' PLRP resins. Alternatively,ethanol precipitation, diafiltration, or gel filtration may be usedfollowed by lyophilization or solvent evaporation under vacuum incommercially available instrumentation such as Savant's Speed Vac.Optionally, small amounts of the nucleic acids may beelectrophoretically purified using polyacrylamide gels.

Lyophilized or dried-down preparations of nucleic acids can be dissolvedin pyrogen-free, sterile, physiological saline (i.e., 0.85% saline),sterile Sigma water, and filtered through a 0.45 micron Gelman filter(or a sterile 0.2 micron pyrogen-free filter). The described nucleicacids may be partially or fully substituted with any of a broad varietyof chemical groups or linkages including, but not limited to:phosphoramidates; phosphorothioates; alkyl phosphonates; 2′-O-methyl;2′-modified RNA; 2′ methoxy ethoxy; morpholino groups; phosphate esters;propyne groups; or chimerics of any combination of the above groups orother linkages (or analogues thereof).

A variety of standard methods can be used to purify the presentlydescribed antibacterial nucleic acids. In brief, the antibacterialnucleic acids of the present invention can be purified by chromatographyon commercially available reverse phase media (for example, see theRAININ Instrument Co., Inc. instruction manual for the DYNAMAX®-300A,Pure-DNA reverse phase columns, 1989, or current updates thereof, hereinincorporated by reference) or ion exchange media such as Waters' ProteinPak or Pharmacia's Source Q (see generally Warren and Vella, 1994,“Analysis and Purification of Synthetic Nucleic Acids byHigh-Performance Liquid Chromatography”, in Methods in MolecularBiology, vol. 26; Protocols for Nucleic Acid Conjugates, S. Agrawal, ed.Humana Press, Inc., Totowa, N.J.; Aharon et al., 1993, J. Chrom.698:293-301; and Millipore Technical Bulletin, 1992, Antisense DNA:Synthesis, Purification, and Analysis). Peak fractions can be combinedand the samples concentrated and desalted via alcohol (ethanol, butanol,isopropanol, and isomers and mixtures thereof, etc.) precipitation,reverse phase chromatography, diafiltration, or gel filtration.

A nucleic acid is considered pure when it has been isolated so as to besubstantially free of, inter alia, incomplete nucleic acid productsproduced during the synthesis of the desired nucleic acid. Preferably, apurified nucleic acid will also be substantially free of contaminantswhich may hinder or otherwise mask the antibacterial activity of theoligonucleotide. A purified nucleic acid, after acidification by one ofthe disclosed methods or by any other method known to those of skill inthe art, is a protonated/acidified nucleic acid that has been isolatedso as to be substantially free of, inter alia, excessprotonating/acidifying agent. In general, where a nucleic acid is ableto bind to, or gain entry into, a target cell to modulate aphysiological activity of interest, it shall be deemed as substantiallyfree of contaminants that would render the nucleic acid less useful.

In particular embodiments, the nucleic acids of the invention arecomposed of one or more of the following: partially or fully substitutedphosphorothioates, phosphonates, phosphate esters, phosphoroamidates,2′-modified RNAs, 3′-modified RNAs, peptide nucleic acids, propynes oranalogues thereof. The nucleic acids may be completely or partiallyderivatized by a chemical moiety including, but not limited to,phosphodiester linkages, phosphotriester linkages, phosphoramidatelinkages, siloxane linkages, carbonate linkages, carboxymethylesterlinkages, acetamidate linkages, carbamate linkages, thioether linkages,bridged phosphoramidate linkages, bridged methylene phosphonatelinkages, phosphorothioate linkages, methylphosphonate linkages,phosphorodithioate linkages, morpholino, bridged phosphorothioatelinkages, sulfone internucleotide linkages, 3′-3′ linkages, 5′-2′linkages, 5′-5′ linkages, 2′-deoxy-erythropentofuranosyl, 2′-fluoro,2′-O-alkyl nucleotides, 2′-O-alkyl-n(O-alkyl) phosphodiesters,morpholino linkages, p-ethoxy oligonucleotides, PNA linkages,p-isopropyl oligonucleotides, or phosphoramidates.

Protonated/Acidified Nucleic Acids

Subsequent to, or during, the above synthesis and purification steps,protonated/acidified forms of the described nucleic acids can begenerated by subjecting the purified, or partially purified, or crudenucleic acids, to a low pH, or acidic, environment. Purified or crudenucleic acids can be protonated/acidified with acid, including, but notlimited to, phosphoric acid, nitric acid, hydrochloric acid, aceticacid, etc. For example, acid may be combined with nucleic acids insolution, or alternatively, the nucleic acids may be dissolved in anacidic solution. Excess acid may be removed by chromatography or in somecases by drying the nucleic acid.

Other procedures to prepare protonated nucleic acids known to theskilled artisan are equally contemplated to be within the scope of theinvention. Once the nucleic acids of the present invention have beenprotonated they may be separated from any undesired components like, forexample, excess acid. The skilled artisan would know of many ways toseparate the oligonucleotides from undesired components. For example,the oligonucleotide solution may be subjected to chromatographyfollowing protonation. In a preferred embodiment, the oligonucleotidesolution is run over a poly(styrene-divinyl benzene) based resin (e.g.,Hamilton's PRP-1 or PRP-3 and Polymer Lab's PLRP) following protonation.

The protonated/acidified nucleic acids can be used directly, or in apreferred embodiment, processed further to remove any excess acid andsalt via precipitation, reverse phase chromatography, diafiltration, orgel filtration. The protonated/acidified oligos can be concentrated byprecipitation, lyophilization, solvent evaporation, etc. When suspendedin water or saline, the acidified nucleic acid preparations of theinvention typically exhibit a pH of between 1 and 4.5 depending upon thelevel of protonation/acidification, which can be determined by how muchacid is used in the acidification process. Alternatively, nucleic acidscan be protonated by passage over a cation exchange column charged withhydrogen ions.

Acid and Nuclease Resistant Nucleic Acids

Generally, nucleic acid preparations near pH 2 to 1 demonstrate betterantibacterial activity than nucleic acids at or near pH 4.5. Many oligobackbones are not stable at pH 2 and experience depurination, although anumber of backbones are relatively stable at a pH of 4 to 5. It has beendiscovered that 2′-O-alkyl, 3′-O-alkyl, and 2′-O-alkyl-n(O-alkyl)nucleic acids are stable at the desired pH of 2 to 1.

In one embodiment, the invention uses nucleic acids that aresubstantially nuclease resistant. This includes nucleic acids completelyderivatized by phosphorothioate linkages, 2′-O-methylphosphodiesters,2′-O-alkyl, 2′-O-alkyl-n(O-alkyl), 2′-fluoro,2′-deoxy-erythropentofuranosyl, p-ethoxy, morpholino nucleic acids,p-isopropyl nucleic acids, phosphoramidates, chimeric linkages, and anyother backbone modifications, as well as other modifications, whichrender the nucleic acids substantially resistant to endogenous nucleaseactivity. Additional methods of rendering nucleic acids nucleaseresistant include, but are not limited to, covalently modifying thepurine or pyrimidine bases that comprise the nucleic acid. For example,bases may be methylated, hydroxymethylated, or otherwise substituted(e.g., glycosylated) such that the nucleic acids comprising the modifiedbases are rendered substantially nuclease resistant.

Although 2′-O-alkyl substituted nucleic acids and molecules with similarmodifications exhibit marked acid stability and endonuclease resistance,they are sensitive to 3′ exonucleases. In order to enhance theexonuclease resistance of 2′-O-alkyl substituted nucleic acids, the 5′and 3′ ends of the ribonucleic acid sequence are preferably attached toan exonuclease blocking function. For example, one or morephosphorothioate nucleotides can be placed at either end of theoligoribonucleotide. Additionally, one or more inverted bases can beplaced on either end of the oligoribonucleotide, or one or more alkyl,e.g., butanol-substituted nucleotides or chemical groups can be placedon one or more ends of the oligoribonucleotide. An enzyme-resistantbutanol preferably has the structure CH₂CH₂CH₂CH₂—OH (4-hydroxybutyl)which is also referred to as a C4 spacer. Accordingly, a preferredembodiment of the present invention is a protonated/acidified nucleicacid comprising an antibacterial nucleic acid having the followingstructure:

A-B-C

wherein “B” is a 2′-O-alkyl or 2′-O-alkyl-n(O-alkyl) oligoribonucleotidebetween about 1 and about 98 bases in length, and “A” and “C” arerespective 5′ and 3′ end blocking groups (e.g., one or morephosphorothioate nucleotides (but typically fewer than six), invertedbase linkages, or alkyl, alkenyl, or alkynl groups or substitutednucleotides). A partial list of blocking groups includes inverted bases,dideoxynucleotides, methylphosphates, alkyl groups, aryl groups,cordycepin, cytosine arabanoside, 2′-methoxy-ethoxy nucleotides,phosphoramidates, a peptide linkage, dinitrophenyl group, 2′- or3′-O-methyl bases with phosphorothioate linkages, 3′-O-methyl bases,fluorescein, cholesterol, biotin, acridine, rhodamine, psoralen andglyceryl.

Wound Treatment

The devices of the invention can be used in the management and care ofany of a variety of wounds and lesions, including but not limited toabrasions, burns, lacerations, puncture wounds, bites, chronic fungatinglesions, chronic pressure ulcers, traumatic wounds and the like. Thedevices of the invention are useful in the closure and protection ofthese wounds, and to help promote wound management and the healingprocess. The particular device of the invention to be used for any givenwound will depend on the nature and extent of the wound, as will beapparent to one skilled in the art upon reading the present disclosure.

Wound Dressings

The first embodiment of the invention provides a dressing comprised of aflexible solid substrate and protonated/acidified nucleic acids intendedfor use as a temporary dressing on burns, wounds and other lesions. Thedressing forms a barrier against bacterial or other contamination. Thedressing preferably remains flexible and facilitates movement, promotingearly physical therapy. The nucleic acid formulations may be impregnatedinto the dressing, or may be a coating on the dressing, with the coatingon the side to lie adjacent to the patient. Types of wound caredressings encompassed by the invention include, but are not limited to,alginates, composits, exudate absorbers, foams, gauzes, hydrocolloids,and hydrogels. Exemplary bandages for use with the present inventioninclude, but are not limited to, those described in U.S. Pat. Nos.5,718,674, 5,692,937, 5,499,966, 5,376,067, 4,867,821, 4,672,956,4,655,202 and 4,377,159.

The dressing of the invention may be formed from any material known inthe art, including biologically derived materials and syntheticmaterials. Preferably, the flexible solid substrate is a syntheticmaterial, and more preferably a woven synthetic material in the form ofa mesh. In a preferred embodiment, the flexible substrate is amultifilament or monofilament polyester mesh sheet. In another example,a sponge or other substrate may replace the mesh netting, wheremedically appropriate and if its properties match the desired end. Theprotonated/acidified nucleic acids are applied to the substrate of thedressing, e.g., a fibrous mesh netting, as an aqueous solution anddehydrated.

The dressing may be used directly, or may be adhered to a backing, e.g.,a self-adhesive backing. Such backing is preferably of a flexiblematerial, and even more preferably has an adhesive on the backingsurrounding the dressing to allow self-adhesion of the bandage. In oneexample, the backing is a flexible strip having a coating of adhesivedeposited on at least the lower planar surface of the strip. A dressingpad of the invention is attached to the lower planar surface of thestrip and centered such that a portion of the adhesive strip extendsfrom each end of the wound pad. The wound pad and strip are die cut in apredetermined shape, thereby separating the wound pad and strip into anouter surrounding frame and inner bandage. Such bandages are describedin U.S. Pat. Nos. 5,792,092 and 5,685,833, which are incorporated hereinby reference.

The dressing of the invention can also be separate and held in place bythe elastic forces of a bandage, e.g., a gauze coated withprotonated/acidified nucleic acids held in place by an elastic bandage.Elastic bandages for use in the invention preferably have good elasticproperties which can be uniform over the width of the bandage. Thefabric may be woven or preferably non-woven. The use of a non-wovenfabric in elastic bandages of the invention can provide a desirabletextile ‘feel’ to the surface of the bandage. Additionally use of anabsorbent non-woven fabric can provide the bandage with a degree ofabsorbency for water and body fluids such as blood. In one example, anelastic bandage can be used which comprises an inner layer of fabric andan outer layer of fabric bonded to a central layer, such as is describedin U.S. Pat. No. 4,414,970.

A vapor permeable film of plastic material occlusive to moisture andbacteria may additionally be joined to one side of the impregnated meshnetting to form an external surface of the dressing. The cast dressingis then cut to the desired size of individual dressings.

The protonated/acidified nucleic acids of the invention may be added inan amount that allows effective dissemination of the biocidal activityfrom the adhesive preparation.

Sutures

Sutures are often used in the closing of a wound, and currently suturingis the method of choice for closing most surgical wounds. The type ofsuture used will vary depending on the type and extent of the wound, thetissue involved, and a particular patient's healing ability.

Sutures within the scope of this invention can be of any type used orcontemplated for use in wound closure. The suture can be synthetic ornatural, absorbable or nonabsorbable, or a monofilament or multifilamentin a braided, twisted or covered form. In addition, the sutures can beattached to one or more needles, if desired. Examples of absorbablemonofilament sutures include natural sutures such as surgical gut andcollagen, and synthetic sutures such as homopolymers and copolymers ofp-dioxanone. Examples of absorbable multifilament sutures includesutures prepared from fiber-forming polymers of one or more lactones,e.g., Vicryl.®. poly(lactide-co-glycolide) multifilament suture.Examples of nonabsorbable monofilament and multifilament sutures includenylon, polypropylene, steel, polyvinylidene fluoride, linen, cotton,silk, and polyesters such as polyethylene terephthalate (PET). Thepreferred sutures are nonabsorbable, multifilament sutures, preferablypolyester sutures. The most preferred suture is PET.

The protonated/acidified nucleic acids of the invention may be added inan amount that allows effective biocidal activity from the coating.Generally, the protonated/acidified nucleic acid is used in aconcentration of 0.5 to 40%, more preferably 1.0 to 20%, even morepreferably between 5% to 10%.

Adhesives

The present invention includes an adhesive compound which incorporatesan adhesive component containing a protonated/acidified nucleic acidpreparation. The protonated/acidified nucleic acids are preferablyhomogeneously dispersed throughout the adhesive layer. Activeprotonated/acidified nucleic acids of the present compositiondisassociate from the surface or leach out of the adhesive matrix overtime, delivering biocidal activity at a distance from the adhesivesurface.

The adhesive of the present invention is specifically suited for use inskin contact applications during and after medical procedures, forexample, as an adhesive in surgical drapes, wound dressings and tapes.The preferred adhesive composition incorporates acrylic polymers andadded tackifiers to form an adhesive which is particularly suited foruse in medical procedures.

A preferred combination of acrylic polymers to form the adhesivecomposition includes the combination of a low molecular weight solidacrylic polymer and a medium molecular weight solid acrylic polymer in aratio of about 1 to 4, respectively, to optimize the adhesion of theadhesive to skin, cohesion and resistance to cold flow. A low molecularacrylic polymer is a polymer having a molecular weight ranging fromabout 90,000 to about 120,000, while a medium molecular weight acrylicpolymer has a molecular weight ranging from about 140,000 to about160,000. Suitable low molecular weight solid acrylic polymers and mediummolecular weight solid acrylic polymers are available from SchenectadyInternational, Inc. under Product Nos. HRJ-4326 and HRJ-10127,respectively.

The adhesive component of the composition can also include tackifiers asare well known in the art. Tackifiers contemplated include SYLVATEC,ZONAREZ and FORAL which are available from Arizona Chemical andHercules, Inc.

The protonated/acidified nucleic acids of the invention may be added inan amount that allows effective dissemination of the biocidal activityfrom the adhesive preparation. Generally, the protonated/acidifiednucleic acid is used in a concentration of 0.5 to 40%, more preferably1.0 to 20%, even more preferably between 5 to 10%.

Wound Sealant

In yet another embodiment of the invention, a wound sealant comprisingprotonated/acidified nucleic acids are used to aid in wound closure.Wound sealants can be used alone or with additional help from otherclosing devices or methods. For example, wound sealants can be used inconjunction with sutures, adhesive tape, bandages, and the like toimprove wound closure integrity. Wound sealant can also be used alone insituations involving coagulopathy, friable tissues, adhesions that causebleeding when sutures would be ineffective to control the bleeding, andthe like. Other potential uses of wound sealants of the inventioninclude sealing vascular suture lines, reinforcing pulmonary andesophageal staple lines and fixing split-thickness skin grafts. SeeSpotnitz et al., Wound Healing, 77:651-669 (1997).

One example of a wound sealant is fibrin sealant, which is comprised offibrinogen and a fibrinogen activator such as thrombin and batroxobin.The fibrinogen activator can be present in various concentrationsdepending on the desired time to form a clot. When the fibrinogenactivator is thrombin, at thrombin concentrations greater than 100 unitsper ml or so in the wound sealant, the fibrinogen concentration becomesthe rate limiting step in coagulation. At concentrations lower thanabout 100 μ/ml, the thrombin level is the rate controlling substance inthe wound sealant. Thus, thrombin concentration can be used to controlthe time to gelation.

Another example of a wound sealant is a platelet glue wound sealantcomprising a plasma-buffy coat concentrate as described in U.S. Pat. No.5,733,545. This sealant contains platelets, fibrinogen, and a fibrinogenactivator in a concentration sufficient to initiate clot formation.

The protonated/acidified nucleic acids of the invention may be added inan amount to effectively treat and/or prevent infection in situs of awound. Generally, the protonated/acidified nucleic acid is used in aconcentration of 0.5 to 40%, more preferably 1.0 to 20%, even morepreferably between 5 to 10%.

Skin Substitutes

Another embodiment of the invention provides skin substitutes comprisingprotonated/acidified nucleic acids. Skin substitutes are commonly usedas dressings, especially for burn victims. They can be used to maintaina clean wound environment until skin grafting can be achieved, or may bea dressing placed on a partial-thickness wound. In another embodiment ofthe invention, substitute skin dressings are provided withprotonated/acidified nucleic acids either coated on the surface to beplaced adjacent to the patient, or interspersed throughout the skinsubstitute. For a review of such skin substitutes, see Staley et al.,Adv. Wound Care 10:39-44 (1997).

Biological dressings that may be used in the invention fall into threecategories: heterografts or xenografts (e.g., pig skin), homografts orallografts (e.g., cadaver skin) and amnion (placenta). These dressingsmay be coated on the surface intended to contact the patient with aneffective amount of the modified nucleic acids of the invention.

Preferably, the skin substitutes of the invention are biosyntheticdressings. These include: Biobrane, a flexible nylon fabric impregnatedwith collagen and bonded to a silicone membrane; collagen derivativessuch as SkinTemp, Medifil, Kollagen, which are typically formed fromanimal collagen; EZ-Derm, a pigskin impregnated with the preservativealdehyde; and Alginates, which are derived from seaweed and releasecalcium ions to help with homeostasis. These dressings may be coatedwith the protonated/acidified nucleic acids of the invention and/or havethe modified nucleic acids impregnated into the fiber of the dressing.

Cultured skin substitutes may also be used in the present invention.These include: cultured epidermal autografts, which are produced from apatient's own keratinocytes; Dermagraft, having a collagen base withhuman neonatal fibroblasts injected into the matrix; Composite skin,Graft skin, a bilayered cultured skin containing human fibroblasts on abovine collagen lattice; Alloderm, an allograft dermis with all immunecells removed; and Integra, a bovine collagen dermis with an outersilicone membrane layer.

Other similar skin substitutes can also be used, as will be apparent toone skilled in the art upon reading this disclosure.

The skin substitute may be impregnated with the protonated/acidifiedoligonucleotide, or it may be coated on the side that will contact thepatient. The solution used for impregnation or coating may be in anyconcentration, but is preferably 0.1 to 40% nucleic acid, morepreferably 1.0 to 20%, even more preferably between 5 to 10%.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g., amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isaverage molecular weight, temperature is in degrees Centigrade, andpressure is at or near atmospheric.

Example 1 Protonation/Acidification of Nucleic Acids

Protonated/acidified forms of the described nucleic acids can begenerated by subjecting purified, partially purified, or crude nucleicacids, to a low pH (e.g., acidic) environment. Purified or crude nucleicacids were protonated/acidified with acid chosen from a group includingphosphoric acid, nitric acid, hydrochloric acid, and acetic acid.

Pooled fractions of a strong anion exchange (SAX)-purifiedoligonucleotide (at approximately 2-25 A₂₆₀ per ml) were pumped onto aPRP (Hamilton Co.) column. This was followed immediately with an excessof dilute acid (e.g., 25 mM HCl) until the eluent was acidic. The columnwas then washed with purified water (no salt or buffers) until theconductivity and pH of the eluent returned to essentially backgroundlevels. The oligonucleotide was then dried down in a commerciallyavailable vacuum evaporator. Alternatively, the oligonucleotide wassuspended in dilute acid and either chromatographed over the PRP orsimilar column as described above, or chromatographed over a sizeexclusion column (e.g., BioRad Biogel P2 or P4) using water as solvent.Alternatively, a desalted nucleic acid may be dissolved in alkaline saltsolution (e.g., 0.4 M NaCl and pH 12, 25 mM NaOH), run on a PRP column,washed with acid followed by water, and then eluted, as described above.Alternatively, a nucleic acid may be chromatographed over a cationexchange column that is in the H+ form, collected and dried down asdescribed above.

Nucleic acids were also acidified by adding an acid, e.g., HCl (0.1 N),directly to a nucleic acid solution (approximately 300 A₂₆₀ per ml)until the pH of the solution reached pH 1 to pH 3. The acidified nucleicacids can then be run over an acid stable size exclusion column such asa BioRad Biogel P2 or P4 column.

Lyophilized or dried-down preparations of nucleic acids to be used inbacterial experiments were dissolved in pyrogen-free, sterile,physiological saline (i.e., 0.85% saline), sterile Sigma water, andfiltered through a 0.45 micron Gelman filter (or a sterile 0.2 micronpyrogen-free filter prior to animal studies).

When suspended in water or saline, the nucleic acid preparationstypically exhibited a pH between 1 and 4.5 depending upon the level ofprotonation/acidification, which is determined by how much acid is usedin the acidification process.

Example 2 Bacterial Growth Studies

The efficacy of the protonated/acidified nucleic acids of the inventionis shown in the following bacterial growth studies.

Limited Nutrient Growth Study

For the limited nutrient growth study, cells were taken off plates andsuspended in PBS to give a final concentration of 10⁵ CFU/ml and a finalvolume of 1 ml. Mueller-Hinton broth was added (40 μl for S. aureus ACC# 13301, 20 μl for P. aeruginosa ACC # 10145). 100 μl of water or 100 μlof nucleic acid (32 A₂₆₀ units, 2′-O-methyl ribonucleotides,phosphodiester linkage, 5′ and 3′ inverted T end-blocked, sequenceCGCCATTGG, SEQ ID NO: 1) was added and the tubes were incubated at 35°C. without shaking for approximately 24 hours. The A₆₂₅ was measured andthe percent inhibition calculated as a percent of the control. Theresults are in the following table:

Bacteria pH of Nucleic Acid Inhibition of Growth (%) S. aureus WaterControl-pH 7 0 S. aureus 2 100 S. aureus 3 100 S. aureus 4 100 S. aureus5 16 S. aureus 7 0 P. aeruginosa Water Control-pH 7 0 P. aeruginosa 2100 P. aeruginosa 3 100 P. aeruginosa 4 100 P. aeruginosa 5 0 P.aeruginosa 7 0

Stationary Growth Study

A stationary growth assay was also performed to study the effect of pHon the anti-bacterial activity of nucleic acids. Cells were taken offplates and suspended in saline to give a final concentration of 10⁷CFU/ml of S. aureus in 1 ml of PBS. 100 μl of water or 100 μl of nucleicacid (32 A₂₆₀ units, 2′-O-methyl ribonucleotides, phosphodiesterlinkage, 5′ and 3′ inverted T end blocked, sequence CGCCATTGG, SEQ IDNO: 1) was added and the tubes were incubated at 35° C. without shakingfor approximately 24 hours. Aliquots were plated directly or afterdilutions and incubated at 37° C. and colonies counted after 24 hours.The results are in the following table:

Bacteria pH of Nucleic Acid CFU/ml S. aureus Water Control-pH 7 10⁷ S.aureus 2 0 S. aureus 3 10³ S. aureus 4 10⁶ S. aureus 5 10⁷ S. aureus 710⁷

From these results, it was concluded that lowering the pH of anucleotide conferred upon it bactericidal and bacteriostatic effects.Next, the effect of sequence identity and length were explored.

Example 3 In Vivo Efficacy of Protonated/Acidified Olignonucleotides

Efficacy in Treatment of Strep. pyogenes Skin Infection on a Dog

A 135 lb. 2 year old, female Newfoundland had sustained a cut on herabdomen and developed a Strep. pyogenes infection. The area was swollen,inflamed and painful to the touch Treatment with Neosporin®(Warner-Lambert, Co.) for 3 days failed to produce any improvement. Twotreatments, separated by 12 hours, directly to the injury with2′-O-methyl substituted ribonucleotides that were phosphodiester linked,pH 1.5, end blocked with butanol at both the 5′ and 3′ ends, withsequence of ACGCGCCATTAT (SEQ ID NO: 2), completely cleared up theinfection, swelling, inflammation, and sensitivity to touch.

Example 4 Protonated/Acidified Nucleic Acid Efficacy in a TopicalPseudomonas Burn Model of Infection

Protonated/acidified nucleic acids were evaluated for their in vivotherapeutic efficacy for the treatment of burn wound infection caused byPseudomonas aeruginosa. A burn wound infection model was established inmice using a highly pathogenic burn wound clinical isolate of P.aeruginosa. Lethality doses of the bacteria were determined for tworoutes of infection (subcutaneous and topical), representing systemicand local forms of infection.

Animals

Six week old BALB/c female mice were obtained from the mouse breedingcolony at DRES, with breeding pairs purchased from Charles River CanadaLtd. (St. Constant, Quebec, Canada). The use of animals described inthis study was approved by DRES Animal Care Committee. Care and handlingof animals described in this study followed guidelines set out by theCanadian Council on Animal Care.

Using this infection model, treatment with protonated/acidified nucleicacids using various routes of drug administration was evaluated andoptimized. The protonated/acidified nucleic acids tested are summarizedin the following table:

Protonated/acidified Nucleic Acids Chemical Structure B2HO—CH₂CH₂CH₂CH₂-ACg•CgC•CAU•Ugg-CH₂CH₂CH₂CH₂—OH pH (1.5-2.0) B7HO—CH₂CH₂CH₂CH₂-ACg•CgC•CAU•Ugg-CH₂CH₂CH₂CH₂—OH pH(~4.5) BUBdihydroxydibutyldiphophate 2′-O-methyl uridine pH (1.5-2.0) C2dibutyl-diphosphate 2′-O-methyl uridine 2HO—CH₂CH₂CH₂CH₂-AUG-CH₂CH₂CH₂CH₂—OH 3 HO—CH₂CH₂CH₂CH₂-G-CH₂CH₂CH₂CH₂—OH4 HO—CH₂CH₂CH₂CH₂—UUU—CH₂CH₂CH₂CH₂—OH 5HO—CH₂CH₂CH₂CH₂-GGG-CH₂CH₂CH₂CH₂—OH 6 2′-O-methyl Uridine monophosphate7 HO—CH₂CH₂CH₂CH₂-CgC•CAU—CH₂CH₂CH₂CH₂—OH

Bacteria

Pseudomonas aeruginosa (Strain Utah 4) was initially cultured on thetripticase soya broth, aliquoted, and frozen at 70° C. Prior to use,aliquots were thawed and diluted serially in sterile PBS just prior toadministration into animals. To ensure viability and virulence, aliquotsof the bacteria were periodically re-amplified in tripticase soya brothand colonies determined on tripticase soya agar plates.

Establishment of Burn Wound Infection

Burn wound infection in mice can be established by subcutaneous ortopical administration of the bacteria to the sites of the burn. LD₅₀values were determined using the method of Reed and Muench, and werefound to be approximately 4×10⁸ and 2×10⁹ CFUs, respectively, forsubcutaneous and topical routes of infection. These lethal dosages ofthe P. aeruginosa strain used were found to change during the course ofthis study due to possible decreases in bacterial viability andvirulence during storage. As a result, these values were regularlyre-checked and adjusted. For all treatment studies, approximately 5 LD₅₀of the bacteria were used. The survival pattern of the mice infectedwith 5 LD₅₀ of the bacteria administered by these two routes ofinfection was similar (FIG. 1). Both routes of administration resultedin eventual death of all mice in the test groups by day 3 postinfection. All control animals which received equivalent doses ofbacteria by either subcutaneous or topical administration without theburn were asymptomatic and found to be completely resistant to theinfection, In the mice that received the burn and infection, the LD₅₀ ofthe bacteria administered topically was approximately 5-fold higher thanthe subcutaneous route. Unless otherwise stated, all treatment studiesdescribed below were carried out using the subcutaneous route ofinfection. This route of administration was chosen for subsequentstudies as it does not require pretreatment of the mice withcyclophosphamide at three days prior to infection, and it causes astronger systemic infection.

For establishing the lethal doses of the bacteria for the systemic burnwound infection, groups of mice were anesthetized with ketamine/xylazinemixture (50 mg/kg each, given intramuscularly), their backs were thenshaved using a clipper, razor and shaving cream. To induce a burn in theback of these animals, a brass bar (10×10×100 mm) was heated in boilingwater for 15 minutes. The end of the heated bar was then applied on theshaved back of the mice for 45 seconds. After a waiting period of 30minutes, 50 μl of the bacterial inoculum (containing approx. 1×10⁸⁻¹¹CFU of total bacteria) was then applied subcutaneously into the sites ofthe burn on the animal back. The mice were then allowed to recover andwere monitored daily for symptoms and deaths. For establishment of atopical infection, the mice were pre-primed with cyclophosphamide (200mg/kg body weight, i.p.). Three days later, the mice were shaved andburns were induced as described above. The inoculum, containing the samenumbers of bacteria, was then topically applied (100 μl) evenly on thesites of the burn, and a custom made “mouse jacket” was then put on theinfection site, for at least 2 hours. These mice were then monitoreddaily for symptoms and deaths.

Treatment of Burn Wound Infection

To determine the effectiveness of various protonated/acidified nucleicacids for the treatment of burn wound infection, mice weresubcutaneously or topically infected with 5 LD₅₀ of P. aeruginosa asdescribed above. Mice were then treated in the following manner. Fortreatment of systemic infection (infection by subcutaneous injection ofthe bacteria), mice were treated at 2 and 8 hours post infection on day1, and twice daily on days 2 and 3. Treatment with variousprotonated/acidified nucleic acids was administered subcutaneously,intravenously and/or topically. For topical treatment of burn woundinfection, mice were treated on the same schedule as onintravenous/subcutaneous treatment. The concentrations of theprotonated/acidified nucleic acids were 335-360 A/ml for subcutaneous(volume 200 μl) and intravenous (volume 100 μl) administrations, 1800A/ml for topical administration (50 μl).

Bacterial Determination of Organ Homogenates

To determine the bacterial load in the blood and organs of experimentalanimals, blood, spleens, livers and the burnt skins were asepticallyremoved. The blood (100 μl) was serially diluted in sterile PBS and 100μl of the diluted blood was plated for growth in tripticase soya agarplates. For the organs, they were homogenized in 2 ml (spleens andskins) or 5 ml (livers) of sterile PBS using a hand-held tissue grinder.The tissue homogenates were serially diluted in sterile PBS, plated forgrowth in TSA, and the inoculated plates were incubated at 37° C.overnight. The number of CFUs was then determined by the presence ofcolonies on these plates.

Statistics

The survival rates of control and treated mice were compared using theMann-Whitney impaired nonparametric one-tailed test. These tests wereperformed using the GraphPad Prism software program (version 2.0;GraphPAD Software, Inc., San Diego, Calif.). Differences were consideredstatistically significant at p<0.05.

Optimization of Routes of Administration

To determine the most effective route(s) of administration for theoligonucleotides, mice which were systemically infected were treatedwith protonated/acidified B2 by the subcutaneous and intravenous routes(FIG. 2). Using both subcutaneous and intravenous routes to treat micewas found to be the most efficacious, resulting in 100% survival rate(p<0.01 vs control). When treatment was administered by subcutaneous orintravenous route alone, the efficacies were 40% (p>0.05 vs control) and80% (p<0.05 vs. control), respectively. These results indicate thatusing both subcutaneous and intravenous administrations of theprotonated/acidified nucleic acids provides optimal therapeuticeffectiveness against systemic burn wound infection.

A total of 12 protonated/acidified oligonucleotides were tested and ofthese, two, B2 and U, were found to be extremely efficacious in the postexposure treatment of burn wound infection (90-100% survival rates vs.0% for untreated control, p<0.01). The comparative efficacy of theseprotonated/acidified nucleic acids in all studies is summarized below inthe following table:

Protonated/acidified # Survivors/Total p < Infected Nucleic AcidsAnimals Tested % Survival Control No Treatment Control  1/45 2 — B228/30 93 <0.0001 B7 2/5 40 0.0867 BUB 7/8 87.5 <0.0001 C2 17/18 94<0.0001 2 3/5 60 0.0184 3 3/5 60 0.0184 4 2/5 40 0.0867 5 3/5 60 0.01846 0/5 0 >0.05 7 2/5 40 0.0867

Protonated/acidified nucleic acids differ greatly in their therapeuticeffectiveness against burn wound infection, ranging in this study from0% (6) to 94% (C2). In all, C and B2 were the most efficacious, with 94%(17/18 C2) and, with 93% (28/30 B2) of the mice responding to treatment(p<0.0001 compared to control) followed by I rate (p<0.001).Protonated/acidified nucleic acids 2, 3, 5 were moderately efficacious(60% effectiveness), while B7, 4, 7 were marginally effective (40%effectiveness). Protonated/acidified nucleic acid 6 did not appear toshow any therapeutic activity in this study.

These protonated/acidified nucleic acids were effective when giveneither systemically by intravenous and subcutaneous administration, orgiven locally to the affected site in the skin by topical application.Treatment using these two routes resulted in almost 100% survival ratesand complete eradication of the bacteria from infection sites in thelivers, spleens and blood. All untreated control mice died from theinfection, with high numbers of bacteria recovered from their tissues.

Comparison between B2 and Ciprofloxacin

The efficacy of protonated/acidified nucleic acid B2 for the treatmentof burn wound infection was compared to that of ciprofloxacin, a potentfluoroquinolone that has been shown to be efficacious in the treatmentof burn wound infection. Protonated/acidified nucleic acid B2 was foundto be equally efficacious as ciprofloxacin for the treatment of burnwound infection, with both therapeutic agents resulting in 100% survivalrates. The following tables show a comparison of in vivo efficacy of B2and ciprofloxacin for the treatment of burn wound infection.

Survival Rates No. Survivors/Total Antibiotic Animals % Survival p <Control Untreated control 0/5  0 — Ciprofloxacin 5/5 100% <0.01 B2 5/5100% <0.01

Microbiological quantitation of tissues Average CFU Antibiotic BloodLiver Spleen Skin Untreated control 2.3 × 10e⁶ 2.1 × 10e⁷ 3 × 10e⁶ NDCiprofloxacin 1.8 × 10e⁵ NG NG 3 × 10e⁶ B2 1.4 × 10e³ NG NG 2 × 10e⁶Where NG = no growth, ND = no data. Microbiological comparisons of theCFUs in livers, spleens, skin and blood of both treated groups reveal nosignificant difference in the abilities of the drugs to eradicate theseorganisms from these infection sites.

Topical Protonated/Acidified Nucleic Acid Treatment of Burn WoundInfection

To determine whether burn wound infection could be effectively treatedby protonated/acidified nucleic acids administered topically, mice wereinfected by topically applying the bacteria into the burn sites on theanimals' backs, and treated in the same manner as described above. Thefollowing table summarizes the results showing the efficacy of topicallyapplied protonated/acidified nucleic acids for the treatment of burnwound infection induced topically:

Survival Rates Protonated/acidified Protonated/ Nucleic Acid acidifiedConcentration No. Survivors/ % p < Nucleic Acids (A/ml) Total SurvivalControl Untreated —  0/10 0 — Control (PBS) B2 1800  9/10 90 <0.01 B2335 2/5 40 >0.05 B7 1800 2/5 40 >0.05

Microbiological quantitation of tissues Group Mouse # Blood LiversSpleens Skins Untreated 1 ND 5.5 × 10e⁷ NG ND Control 2 ND 1.0 × 10e⁶1.9 × 10e⁸ ND 3 ND 2.4 × 10e⁸ 5.6 × 10e⁹ ND B2 1 NG NG NG 3.9 × 10e⁸(1800 A/ml) 2 NG NG   2 × 10e⁵ 4.2 × 10e⁷ 3 NG NG NG 2.1 × 10e⁹

Protonated/acidified nucleic acid B2 administered topically was found tobe very effective in the treatment of local burn wound infection,resulting in 90% survival rate, while all untreated control animalsinfected topically succumbed to the infection. The effectiveness ofprotonated/acidified nucleic acid administered topically was found to bedependent on drug concentration, decreasing the concentration of theprotonated/acidified nucleic acid B2 from 1800 A/ml to 335 A/ml resultedin a sharp decrease in survival rates from 90% to 40%. When the blood,spleens, livers and skins of 3 mice which were treated with B2 (1800A/ml) were analyzed and compared to that of the untreated controls, itwas found that all blood, spleen and two out of three liver samples fromthe treated group were devoid of any detectable CFUs, in contrast tothat of the untreated group which harbored high numbers of bacteria inthese tissues.

Example 5 Antibiotic Wound Dressing

A polyester woven multifilament mesh netting having a thickness of 0.020inch is synthesized according to the method described in U.S. Pat. No.5,676,967, which is incorporated herein by reference. The netting issynthesized having an average pore size of about 1/32 inch.

An aqueous-based solution of 5% protonated/acidified oligonucleotidehaving the sequence ACGCGCCATTAT (SEQ ID NO: 2) is prepared according tothe method of Example 1. This aqueous solution is coated on the meshnetting and adhered to the fibers of the netting. The coated netting isthen subjected to dehydration at about 40° C. Following dehydration, oneside of the coated netting is thermally bonded to a 1 mm thick solidpolymeric film.

Example 6 Sutures

A solution of protonated/acidified C2 monomer is prepared. The solutioncontains a 10% solution of C2 monomer in an appropriate carrier, such assterile water or phosphate buffered saline. A size 2/0 (USP standard)Mersilene.®. PET braided multifilament suture is coated at roomtemperature with the coating solution using conventional laboratorycoating equipment, and the coated suture is subsequently dried in air atelevated temperature to remove the solvent.

Example 7 Adhesives

The antimicrobial-containing adhesive composition of the presentinvention is manufactured using the following procedure. Acrylicpolymers and tackifiers are thoroughly mixed at a temperature of about121° C. to about 127° C. The adhesive composition includes approximately17% low molecular weight acrylic polymer (HRJ-4326 from SchenectadyInternational, Inc.), 67% medium molecular weight polymer (HRJ-10127from Schenectady International, Inc.) along with 16% FLORAL 105synthetic resin from Hercules, Inc. as a tackifier. Once mixed, thepolymers and tackifiers are heated to approximately 154° C. to 157° C.with continued mixing until uniform, followed by cooling to about 65° C.Protonated/acidified oligonucleotide (pH 1.5, sequence ACGCGCCATTAT, SEQID NO: 2) is then added to the polymer mixture to a final concentrationof 2% and mixed until uniform.

The adhesive composition is then melted and applied to a substrate layerin a thin coating approximately 0.05 mm in thickness. The substrate is aco-polyester surgical drape material such as available from DuPont underthe tradename HYTREL. The drape can include a layer ofprotonated/acidified oligonucleotide applied to the substrate layer on aside opposite that to which the adhesive is applied.

Example 8 Wound Sealant

A unit of blood is withdrawn from a patient in sterile fashion. Theblood is collected in a standard sterile blood donor set, using CPDA-1anticoagulant (Terumo Corporation). The collected blood is connected toa sterile disposable blood processing set manufactured by ElectromedicsInc., which is in turn mounted on an ELMD500 Autotransfusion System alsoby Electromedics. The centrifuge insert (125 ml) is set to rotate at5600 RPM, generating forces within the blood on the order of 2000 G. Theblood is then pumped into the insert at 50 ml per minute.

When the insert is nearly full of packed cells with only a small centralcore of clear plasma remaining, the blood pump is stopped and thecentrifuge speed reduced to 2400 RPM. The exit line leading to theplasma collection bag is closed, the exit line leading to anothercollection bag is opened, and blood flow is reinstituted at 50 ml/min.The blood entering the centrifuge bowl is again fractionated at thelower centrifugal force, causing the erythrocytes and neutrophils toremain in the centrifuge bowl.

The plasma fraction continues to exit the bowl, carrying with it most ofthe platelets, monocytes, and lymphocytes into the second collection bagto produce a plasma-buffy coat mixture. Blood continues to be pumpedinto the bowl such that it is filled with erythrocytes, forcing thecentral plasma column and entrained formed elements into the exit line.Flow continues until the point where erythrocytes first begin to enterthe second collection bag. At that point the blood flow is stopped, theexit line to the bag containing the plasma-buffy coat mixture isclamped, and the centrifuge is stopped.

The plasma collection bag containing the plasma-buffy coat mixture isopened, and the blood pump reversed. The platelet-poor red cells andplasma are directed to a third collection bag. The process is repeateduntil all the whole blood that had been collected is processed. Thereconstituted platelet-depleted whole blood may then be reinfused to thepatient.

A sterile assemblage of commonly available blood bag spikes, stopcocksand tubing is constructed such that the platelet bag is connected to athree way stopcock. A second port of the stopcock leads to a 60 ccsyringe, and the third leg leads to a 0.25 square meter surface areahemoconcentrator manufactured by Minntech Inc., the outlet of which isin turn connected to a Terumo Corporation sterile blood transfer bag.The plasma-buffy coat mixture is then aspirated into the 60 cc syringe.By adjusting the attached stopcock, the flow path between syringe andhemoconcentrator is opened. A vacuum of −400 torr is applied to thedischarge port of the hemoconcentrator, and the plunger of the 60 ccsyringe is compressed to force the plasma through the blood path of thehemoconcentrator over about two minutes to produce a plasma-buffy coatconcentrate.

The vacuum is immediately disconnected, and the empty 60 cc syringe isreplaced by a 20 cc syringe full of normal saline. The saline isimmediately infused into the hemoconcentrator to flush residualplasma-buffy coat concentrate from the device into the receivingtransfer bag to form the final plasma-buffy coat concentrate.Protonated/acidified nucleic acid (pH 1.5, sequence ACGCGCCATTAT, SEQ IDNO: 2) is then added to the plasma-buffy coat concentrate to a finalconcentration of 1%.

While the present invention has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims appended hereto.

1. A wound dressing comprising: a solid substrate; and a formulation ofprotonated/acidified nucleic acids.
 2. The wound dressing of claim 1,wherein the formulation of protonated/acidified nucleic acid is fromabout 0.1 to about 5 percent of the dressing dry weight.
 3. The wounddressing of claim 1, wherein the formulation of protonated/acidifiednucleic acids is a coating on said substrate.
 4. The wound dressing ofclaim 1, wherein the formulation of protonated/acidified nucleic acid isinterspersed in the solid substrate.
 5. The wound dressing of claim 3,further comprising a polymeric film bonded to one side of said coatedsolid substrate.
 6. The wound dressing of claim 1, wherein saidpolymeric film has a thickness of about 0.001 inch+/−about 0.0005 inch.7. The wound dressing of claim 1, wherein the solid substrate comprisesa polyester mesh netting formed of woven multifilament polyester.
 8. Asuture comprising: a pliable solid substrate; and a formulation of aneffective amount of protonated/acidified nucleic acids.
 9. The suture ofclaim 8, wherein the formulation of protonated/acidified nucleic acid isfrom about 0.1 to about 5 percent of the dry weight of the suture. 10.The suture of claim 8, wherein the solid substrate is comprised ofsynthetic materials
 11. The suture of claim 10 wherein the solidsubstrate is a polyester.
 12. The suture of claim 8 wherein the sutureis a nonabsorbable suture.
 13. An adhesive composition having antibioticproperties for skin contact applications comprising: an adhesivepolymer; and an effective amount of protonated/acidified nucleic aciddispersed throughout said polymer.
 14. The adhesive composition of claim13, wherein said adhesive polymer comprises a mixture of a low molecularweight solid acrylic polymer and a medium molecular weight solid acrylicpolymer.
 15. The adhesive composition of claim 13, further comprising aneffective amount of a tackifier.
 16. The adhesive composition of claim13, wherein the concentration of protonated/acidified nucleic acids insaid polymer composition is about 0.1% to about 2% by weight.
 17. Asurgical drape comprising: a sheet of polymeric substrate; a coating ofan adhesive composition of claim
 15. 18. The surgical drape of claim 17,wherein said substrate comprises a sheet of a polyester.
 19. A woundsealant comprising: a fibrinogen activator in a concentration sufficientto initiate clot formation; and an effective amount ofprotonated/acidified nucleic acids.
 20. The wound sealant of claim 19,wherein the fibrinogen activator is selected from the group consistingof thrombin and batroxobin.
 21. The wound sealant of claim 19 furthercomprising fibrinogen.
 22. The adhesive composition of claim 19, whereinthe concentration of protonated/acidified nucleic acids in said woundsealant is about 0.1% to about 10% by weight.
 23. A skin substitutecomprising: a flexible support surface; and an effective amount ofprotonated/acidified nucleic acid.
 24. The skin substitute of claim 23,wherein the protonated/acidified nucleic acid is impregnated into thesupport surface.
 25. The skin substitute of claim 24, wherein theprotonated/acidified nucleic acid is coated onto the support surface.26. The skin substitute of claim 24, wherein the concentration ofprotonated/acidified nucleic acids in said skin substitute is about 0.1%to about 2% by weight.
 27. A method of treatment, comprising: covering awound with the wound dressing of claim
 1. 28. A method of treatment,comprising: closing a wound with the suture of claim
 8. 29. A method oftreatment comprising: covering a wound with the surgical drape of claim17.
 30. A method of treatment, comprising: closing a wound with thewound sealant of claim
 19. 31. A method of treatment, comprising:covering a wound with the skin substitute of claim 23.